MXPA02000190A - Preparation of a therapeutic composition. - Google Patents

Abstract

Product R, a novel therapeutic composition for treating viral infections and stimulating the immune system, comprises nucleotides and peptides that have molecular weights not more than 14 KDa and substantially not more than 8 KDa. The composition has a light absorption spectrum with typical absorption ratios of 1.998 at 260nm/280nm and 1.359 at 260nm/230nm.

Description

PREPARATION OF A THERAPEUTIC COMPOSITION RELATED REQUESTS The present is a request for continuation in part of the application in process of the applicant, serial No. 08 / 735,236, filed on October 22, 1996. This application incorporates the contents of the application serial No. 08 / 735,236, through this reference, in its entirety.

BACKGROUND OF THE I NVENTION 1.- FIELD OF THE INVENTION The present invention relates to an improved method for preparing a therapeutic composition, the product R1, as will be defined hereinafter, which contains peptides and nucleotides.

The components of product R have molecular weights not exceeding 14 kilodalton (KDa). 2. - D ESCRI PCIÓ N D E THE RELATED TECHNIQUE The concept of an antiviral agent composed of peptones, peptides, proteins and n-nucleic acid originated in 1934.

Product R was used as a synonym of RETICULOSE in certain literature. For the purposes of this application, product R and RETICULOSE represent two different products After some years of experimentation, said antiviral agent was modified using bovine serum albumin in combination with peptone and ribonucleic acid, to produce an antiviral biotic agent, which is non-toxic, lacks anaphylactogenic properties and is miscible with tissue fluids and the serum of the blood. The agent used will be described as "lipopeptide-nucleic acid compound" and registered under the trademark RETICULOSE® by Chemico Laboratories, Inc .; Physician Desk Reference, page 651, 1960. RETICULOSE® was reported as an antiviral agent to treat a variety of viral infections, such as influenza, herpes, hepatitis A and B. It was then assumed that RETICULOSE® acted as an antiviral agent at least increasing leukogenesis, the synthesis of antibodies and increasing phagocytosis. RETICULOSE® was last sold in the United States in 1964.

The method to prepare RETICULOSE® had been kept as a trade secret by the manufacturer, until the issuance of US Patent 5,849, 10O6, which describes the method of preparing RETICULOSE®.

As described in the U.S. patent 5,849,196, the starting materials for preparing RETICULOSE® consist, by weight, of: 40-50 percent casein; 1 to 10 per , one hundred percent blood albumin; 15 to 40 percent of pept.one of beef; 10 to 25 percent of RNA and 5 to 25 percent of sodium hydroxide. These starting materials are suspended in water, which produces a proportion of proteins (casein, peptone and blood albumin to water equal to about 4.3 to 100 weight percent.) After autoclaving the mixture of the starting materials, the resulting solution is filtered and the pH adjusted to approximately at 8.5 and then at 7.8, after which the neutralized solution is filtered again, the pH is further adjusted to about 7.5 after the solution is diluted.This process produces a mixture of peptides and nucleic acids having molecular weights in the Approximate scale of 1 to 25 KDa.

As taught in U.S. Patent 5,849,196, components of more than 15 KDa of the conventional RETICULOSE® composition are more effective in treating viral diseases, such as HIV, influenza virus, herpes simplex virus, etc. , whereas the components in the approximate scale of 1 to 15 KDa work as inhibitors of phagocytosis.

However, conventional methods suffer from several disadvantages: 1) the method does not guarantee that each preparation produces the final components with the same proportion, so that the product is not reproducible; 2) the conventional method produces a wide scale of final components, which makes it extremely difficult to control the preparation, if possible, because it is necessary to determine too many parameters; 3) the presence of higher molecular weight components, such as a 25 KDa component, essentially the peptides, increases the risks of hypersensitivity or an immunological reaction, and makes the product less stable. Therefore, it is convenient to have a product devoid of the deficiencies of the conventional RETICULOSE®, at the same time that its therapeutic properties are maintained.

BRIEF DESCRIPTION OF THE INVENTION Accordingly, one objective of the present invention is directed to a novel therapeutic composition, the product R. In contrast to the RETICULOSE®, the product R is reproducible, highly stable and non-antigenic. Similar to RETICULOSE®, the product R is a broad-range antiviral agent to treat viral infections, such as infections by human immunodeficiency virus (HIV), herpes simplex virus, adenovirus and papilloma virus. Surprisingly, it has been shown that the product R is effective in stimulating the production of chemokines, including gamma-interferon, interleukin-6 and interleukin-1 (J. Investig. Med. 1996; 44: 347-351), the production of globules. I blood reds (U.S. Patent 5,807,839), the treatment of basal cell carcinoma (U.S. Patent No. 5,902,786) and the treatment of viral infections of canine distemper (U.S. Patent No. 5,807,840).

Another object of the present invention is directed to an improved method for preparing the novel therapeutic composition, product R. According to the improved method herein, Product R comprises novel components that generate a novel UV absorption spectrum and a novel molecular weight profile. In particular product R comprises molecules having molecular weights not exceeding 14 KDa.

Another objective of the present invention is to define the components of the product R, based on physical and chemical methods. Other objects and aspects of the present invention will be apparent from the following detailed description, considered in conjunction with the accompanying drawings. However, it should be understood that the drawings are devised exclusively for illustrative purposes, and not as a definition of the limits of the invention, for which reference should be made to the claims that come at the end. It should be further understood that the drawings are not necessarily drawn to scale and that, unless stated otherwise, they are simply intended to conceptually illustrate the structures and procedures described herein.

BRIEF D ESC RIPTION OF THE DIES In the drawings: Fig. 1 shows an absorption profile or ultraviolet representative of the product R.

Figure 2 shows a representative chromatogram of product R obtained from a reverse phase HPLC analysis.

Figure 3 shows a fractionation profile BioGel P2 of product R.

Figure 4 shows the components of fraction I of the fractionation profile BioGel P-2, resolved in an electrophoresis on a 16 percent SDS-polyacrylamide gel (SDS-PAGE).

Figure 5 shows the relative mass (Mr.) of the two main components of peptide of product R, resolved on a 16% SDS-PAGE.

Figure 6 is a 16% SDS-PAGE, showing the effects of a variety of catabolic enzymes, on product R.

Figure 7 is a flow cytometric histogram, showing the effect of product R on phagocytosis of Dextran-FITC; Y Figure 8 is a flow cytometric hydrograph, showing the effect of product R on phagocytosis of Dextran-BoDipyFL.

DETAILED DESCRIPTION OF CURRENTLY PREFERRED MODALITIES PREPARATION OF THE PRODUCT R In general, the product R is prepared according to the following manner: First of all, the starting materials casein, beef peptone, RNA, BSA and sodium hydroxide are suspended in proportions by weight of: 35-50 percent of casein, 15 to 40 percent of beef peptone; 10 to 25 percent RNA, 1 to 10 percent BSA and 5 to 25 percent sodium hydroxide, in an appropriate volume of distilled water. All materials are generally available, or if not, they can be easily prepared by someone with ordinary experience in the field. While any RNA is suitable for the purpose intended in the present invention, plant RNA is preferred and yeast RNA is most preferred. The ratio of total proteins to volume of distilled water is generally about 1.5-2.5 to 100, by weight; preferably, about 2.2 to 100 by weight. This means that every 1.5 to 2.5 g of total proteins are suspended in about 100 mL of distilled water.

All the starting materials can be obtained generally in the trade, or on the contrary, they can be prepared easily by a person having ordinary experience in the field.

The suspension prepared as mentioned above is then autoclaved at an approximate pressure of 2.27 to 6.81 kg, preferably 3.63 to 4.54 kg, under high temperature, on an approximate scale, for example, 65 to 148 °. C, preferably approximately between 93 ° C and 110 ° C, for a period of approximately 2 to 10 hours, preferably more than 3 hours. As it is known by one who has ordinary experience in the matter, under such conditions, the RNA can be hydrolyzed completely to nucleotides. After autoclaving, the solution is cooled to room temperature, and then allowed to remain at a temperature of 3 to 8 ° C for at least 12 hours, to precipitate the insoluble elements. Alternatively, the cooled solution can be centrifuged at a temperature of less than 8 ° C to eliminate the precipitates.

The resulting solution is then filtered through a 2 micron filter and a 0.45 micron filter, under an inert gas, such as nitrogen or argon, at a pressure of about 6.89 kPa at 41.36 kPa. In a similar manner, the solution is again filtered through a filter that retains the pyrogens, preferably 0.2 microns.

After the above filtration, the solution can be cooled to 3 to 8 ° C again for at least about 12 hours, and it is filtered again in the same manner as described above.

The resulting filtrate is then subjected to analysis to see the total nitrogen content, using methods known to those of ordinary skill in the art, for example, by the method of Kjeldahl, J. G. C. Kjeldahl, Z. Anal. Chem., Volume 22, page 366 (1883), and its improvements. Based on the analysis, the filtrate is then diluted with cold distilled water, to an appropriate volume having a total nitrogen content, preferably between 165 and 210 mg / ml.

The pH of the diluted solution is then adjusted with HCl to a physiologically acceptable pH value, preferably around 7.3 to 7.6, after which the diluted solution is filtered again through a 0.2 miter filter, under a inert gas, as described above.

The product R thus produced contains essentially nucleotides, nucleosides and free nucleic acid bases, of low molecular weight, coming from the complete hydrolysis of the RNA, and small peptides, coming from the partial hydrolysis of the proteins. It is possible that the base hydrolysis of the proteins also produces free amino acids.

It is understood that the use of the filtration technique is essentially to eliminate bacteria or other particles having a size similar to, or greater than, the bacteria. Thus, any filter, regardless of its manufacturer or the material it is made of, is suitable for the intended purpose. All filters used in the present process are widely available to those skilled in the art.

Next, appropriate ampoules are filled with the final filtrate and sealed; for example, 2 ml or 10 ml ampules, under inert gas. The filled ampoules are autoclaved for final sterilization, after which they are ready to be used. ' In use, the product R is administered parenterally or topically to patients who need it, as described in U.S. Patents 5,807,839, 5,807,840 and 5,902,786; whose contents are incorporated here in their entirety, by means of this reference.

CHARACTERIZATION OF THE PRODUCT R The ultraviolet absorption spectrum Figure 1 is a representative ultraviolet absorption spectrum of the product R, measured in a microcube (capacity of 100 μl) of quartz, with path length of 1 cm, using a Shimadzu spectrophotometer, model UV1201 UV-VIS . The product R was diluted with distilled water up to 100 times. The spectrum is recorded between 220-320 nm and shows a maximum absorption at 260 nm and a transit at 235 nm. The absorbance ratio (A) at 260 nm with respect to the absorbance at 280 nm is 1.998 (± 10%), and from A at 260 nm with respect to A at 230 nm it is 1359 (± 10%).

The HPLC profile: Figure 2 is a representative chromatogram of the product R, obtained from a reverse phase HPLC analysis, using a HPLC system Hewlett Packard 1100 (Hewlett Packard Co.), which includes a binary pump (model G1312A ), a diode-forming detector (Model G1315A), a column thermostat (model G 1316A), an autosampler 'with thermostat (model G1329A), a test thermostat and a vacuum degasser (model G 1322A), and a YMC-pack stainless steel column ODS-AQ S-5 μM (YMC, Inc., 3223 Burnt Mill Dr., Wilmington, NC 28403), which has a size of 250 x 10 mm inner diameter and a pore size of 120 angstrom. The mobile phase consisting of a mixture of 0.1M acetic acid: triethylamine is prepared as follows: 6.0 ml of glacial acetic acid is dissolved in 1000 ml of HPLC quality water. The stirred solution of acetic acid is titrated with triethylamine at pH 4.8. The solution is allowed to equilibrate overnight at room temperature and is then filtered through a 52 mm diameter filter and 0.45 μM pore size. The pH of the solution is adjusted again to 4.8, if necessary adding triethylamine before use. The mobile phase is degassed by the vacuum degasser, incorporated in the HPLC flow system. 8 μL of the product R sample is injected by means of an autosampler, to the column that has a temperature setting of 30 ° C for each injection. Then, the column sample is eluted with 0.1M acetic acid: tetylamine as the mobile phase (pH 4.8) at a rate of 1 mL per minute, under a pump pressure of 92-102 bar. The chromatograms (UV absorbances at 260 nm) are operated at 160 minutes per sample, and the data is collected by the diode-forming detector, and then analyzed using the ChemStation HPLC application program from Hewlett-Packard. Graphs are generated and statistical analysis is carried out using the SigmaPlot program. Reverse phase HPLC, under these conditions, results in 13 characteristic HPLC peaks: A, B, C, D, E, F, G, H, I, J, K, L and M, each of which has a characteristic UV absorption profile (data not shown).

The filtration profile in the BioGel P-2: Figure 3 shows a fractionation profile of the product R in a BioGel P-2 column (Bio-Rad Laboratories, Inc.), which has a size of 2.6 cm x 55 cm packed size. After loading the product R in the column the column is eluted with a PBS at 0.1X, preferably PBS of DULBECCO, free of calcium ion (Ca + +) and of magnesium ion (Mg + +), at a flow rate of 0.5 mL per minute. PBS 1X contains 1.47 mM of KH2PO4, 2.67 mM of KCl, 138 mM of NaCl and 8.1 mM of Na2HPO4 7H2O. The eluent passes through a "Uvcord SU" monitor, which is attached to a REC 101 recorder equipped with a 254 nm filter and collected at 12 minutes per fraction in a "Frac 200" fraction collector. Gel filtration chromatography, under these conditions, results in 9 fractions: I, II, II, II, II, III, IV, and IVa. Each individual peak is compared to known nucleotides, known nucleosides and known free nucleic acid bases, eluted at the same or very close volumes, of the respective fractions, as shown in Table I. The known compounds having comparable values are shown in the "Observations" column.

TABLE I The fractions are then concentrated and analyzed by SDS-PAGE (see below) in a 16 percent gel. Staining the gel with silver demonstrates that only fraction I shows essentially two main bands obtainable with silver, which have apparent molecular weights of 4.3 KDa, 5.2 KDa and a band less than 7.6 KDa, as shown in Figure 4.

The relative mass (Mr.): Figure 5 shows the relative mass (molecular weight measurement) of the two main peptide components of the product R, solved in a gel electrophoresis of 16 percent SDS-polyacrylamide (SDS-PAGE) and stained with silver stain, using the "SilverXpress" staining equipment from NOVEX, following the protocol suggested by the manufacturer.

The product R is resolved into two silver-stainable main bands, which have the apparent molecular weight of about 4.3 and about 5.2 KDa. A minor silver stainable component having an approximate molecular weight of 7.6 KDa is also visible in an overloaded SDS-PAGE gel, and there may be oligomeric amounts of other silver-stainable peptides, which have molecular weights ranging from about KDa to around 14 KDa. Coomassie Blue, a universal protein dye, stains the 4.3 KDa band extremely poorly. The three bands, 4.3 KDa, 5.2 KDa and 7.6 KDa, constitute more than about 90 percent of the peptides. In such a manner, the product R consists essentially of molecules having molecular weights below 8 KDa.

Table II shows the amino acid compositions of the 5.2 KDa and 4.3 KDa components. The amino acid analysis of the 5.2 KDa band (sample A) and the 4.3 KDa band (sample B) was carried out on a PE Bio-system 420 analyzer, with automatic hydrolysis, using common chemistry and phenoiso current -thiocyanite (PTIC).

TABLE II Amino Acid Mués, tra A (to Iré dedor Sample B (around 5 2 KDa).% Mol. Of 4.3 KDa). % mol.

Aspartic acid 9.92 8.95 Glutamic acid 19.27 17.30 Serine 1.03 1.23 Glycine 5.74 13.87 Histidine 2.58 3.11 Arginine 0.69 0.52 Threonine 0.73 1.78 Alanine 5.49 8.19 Proline 13.05 15.28 Tyrosine 4.39 3.37 Valine 9.95 5.39. Methionine 2.92 2.21 Isoleucine 5.47 3.45 Leucine 10.99 4.37 Phenylalanine 3.27 1.45 Lysine 5.12 9.53 THE BIOCHEMICAL PROPERTIES OF THE PEPTIDES Some of the biochemical properties of the silver-stainable peptide components of the product R are analyzed; using various catabolic enzymes, in the manner described below: Proteinase K treatment (ICN Biochemicals): Proteinase K is a broad-spectrum, non-specific protease that breaks peptide ligatures at the C-terminus of aliphatic, aromatic and hydrophobic amino acids. It can break all serum peptides completely at 50 μg / ml within one hour. A sample of the product R is incubated in a reaction regulator having 10 mM Tris-HCl, pH 7.6; 0.5% SDS, 1 mM CaCl2, 100 μg / ml proteinase K at 40 ° C for 30 minutes; and then subjected to SDS-PAGE in gel at 16 percent, as described above. Under these conditions the silver staining of the product R shows no significant changes. However, when the amount of proteinase K is increased to 800 μg / mL and the incubation time is extended to one hour, the 5.2 KDa band disappears, but there is no obvious change in the 4.3 KDa band.

Trypsin treatment (Boehrinqer Mannheim, USA): Trypsin is a protease for serine, which specifically breaks the peptide ligatures of lysine and arginine at the C-terminus, at pH 7.5 to 9.0. A sample of product R is incubated in a retention buffer that has 100 mM Tris-HCl, pH 8.0, 0.1 percent SDS and 250 μg / mL of sequence-quality trypsin, at 25 ° C for 19 hours, and then it is subjected to SDS-PAGE in a 16 percent gel. While serum proteins will be broken down to peptides smaller than 4.3 KDa under these reaction conditions, none of the components dyeable with silver, of the product R, is affected by trypsin.

Treatment with chymotrypsin (Boehrinqer Mannheim, USA): Chymotrypsin is a serine protease that specifically hydrolyses the peptide ligatures of tyrosine, phenylalanine and tryptophan in the C terminals. It also breaks the peptide ligatures of leucine, methionine, alanine, aspartic acid and glutamic acid at terminals C, at relatively slower speeds. A sample of product R is incubated in a reaction buffer containing 100 mM Tris-HCl, pH 7.6, 10 mM CaCl2 and 250 μg / ml of sequencing quality chymotrypsin at 25 ° C for 19 hours, and then subjected to to SDS-PAGE in a 16 percent gel. Treatment with chymotrypsin significantly reduces the intensity of the 5.2 KDa and 7.6 KDa bands, but has no apparent effect on the 4.3 KDa band.

Treatment with pronase (Boehrinqer Mannheim, USA): Pronase is a non-specific protease; it acts both on natural proteins and on denatured proteins. It decomposes virtually all proteins to their individual amino acids. The preparation contains various types of endopeptidases, such as serine, and of metalloproteases, exopeptidases, such as carboylpepsidases, neutral protease and neutral and alkaline phosphatases. A sample of product R is incubated in a reaction buffer containing 100 mM Tris-HCl, pH 7.4; 10 mM CaCl2; 0.1% of SDS and 2 mg / ml pronase of S. griseus at 40 ° C for 75 minutes, and then subjected to SDS-PAGE on a 16% gel. All silver stainable components disappear after said pronase treatment.

Treatment with N-qlicosidase F (Boehrinqer Mannheim USA): N-glycosidase F breaks all types of N-glycans bound to asparagine, provided that the amino group and the carboxyl group are present in a peptide ligation, and that the oligosaccharide has the minimum length of the central unit of quitobiosa. A sample of product R is incubated in a reaction regulator containing Dulbecco 0.4X PBS (where PBS 1X contains 1.47 mM KH2PO4, 2.67 mM KCl, 138 mM NaCl and 8.1 mM Na2PO4.7H2O), 0.1% SDS, 0.5% of NP40 and 50 units / mL of recombinant N-glycosidase F, at 37 ° C, for four hours; and subjected to SDS-PAGE in a 16% gel. Treatment with N-glycosidase F does not alter the intensity of any of the R product bands on the 16% SDS gel. Resistance to N-glycosidase F indicates the lack of N-glycan bound to asparagine, which is commonly observed in glycoproteins. The ribonuclease A treatment (ICN Biochemicals, E. U. A.): Ribonuclease A is an endoribonuclease specific for pyrimidine, which acts on single-stranded RNA. A sample of product R is incubated in a reaction regulator that contains 10 mM Tris-HCl, pH 7.4, 3 mM MgCl2 and 1 mg / ml bovine pancreatic ribonuclease A, at 37 ° C for about one hour, and is subjected to SDS-PAGE on a 16 percent gel . Ribonuclease A does not alter the intensity of any of the R product bands resolved by the 16 percent SDS-PAGE gel. Resistance to ribonuclease A excludes the possibility of the presence of an RNA fragment attached to the peptide. The treatment with alkaline phosphatase (Life Technologies, E. U. A.): Calf thymus alkaline phosphatase (CIAP) is a phosphomonoesterase that hydrolyzes the 5'-phosphate groups of DNA, RNA and nucleotides. A sample of product R is incubated in a reaction regulator provided by the manufacturer of the enzyme and 200 units / ml of CIAP at 37 ° C for about one hour, and subjected to SDS-PAGE in a 16 percent gel . CIAP does not alter the intensity of any of the R product bands resolved by SDS-PAGE. A summary of the treatments described above, by the catabolic enzymes, is given in the following Table III, and the results of the treatment are shown in Figure 5, where "-" represents no alteration of the dyeable bands and "+" "represents a substantial alteration of the dyeable bands.

TABLE III Enzyme Sensitivity of the peptide components of the product R (SDS-PAGE) 4.3 KDa 5.2 KDa 7.6 KDa Proteinase K (100 μg / mL) 7 * (800 μg / mL) 7 * Trypsin (250 μg / mL) Chymotrypsin (250 μg / mL) Pronaze (2 mg / mL) N-glycosidase F (50 units / mL) Ribonuclease A (1 mg / mL) Alkaline phosphatase (200 units / mL) * This band it is not clearly identified due to the presence of the enzyme fragments in that region. The complexity of these patterns of enzymatic digestion suggests that the peptide components of the product R can be conjugated with other molecules, such as mononucleotides and / or carbohydrates, or can be inter-linked intra-inter-molecularly. RNA gel electrophoresis: Neither agarose gel electrophoresis nor polyacrylamide gel for nucleic acids generates any dye-stainable band with ethidium bromide, indicating that there are no RNA fragments in product R.

THE EFFECT OF PRODUCT R ON PHAGOCYTOSIS Figures 7 and 8 are flow cytometric histograms representing the fluorescence associated with the cells, showing the effect of the product R on phagocytosis of Dextran-FITC or Dextran-BoDipyFL after 24 hours and 8 days of product treatment R, respectively. The effects of the product R on phagocytosis are tested using a human monocytic cell line, U937. U937 cells are cultured in a medium that has 5 percent R product, or 5 percent PBS, as control, for 24 hours, before the Dextran-FITC test, or for eight days before the Dextran test -BoBipyFL. To measure phagocytosis the cells are continuously fed with a phagocytic marker, such as fluorescently labeled Dextran-FITC, for 5, 15, 30 and 45 minutes, as indicated in Figure 5, or Dextran-BoDipyFL for 5, 15 , 25 and 40 minutes, as indicated in figure 6, at 37 ° C. The amount of a fluorescence associated with the cells is monitored, following phagocytic absorption, using flow cytometric analysis, in accordance essentially with the method described by Sallusto, F. and co-authors (1995), J. Exp. Med., 182 : 389-400, which is incorporated herein by this reference, in its entirety. In these tests, the background values of those corresponding to the experimental samples have been subtracted, and the dead cells have been excluded from the data, using iodide exclusion. propidium Each of FIGS. 7 and 8 shows an unfolding of the logarithmic fluorescence against the number of cells for the PBS control (purple), the product R treatment (green) and the background dextran that binds to the cells (black). The purple curves (PBS control) are substantially overlapped with the green curves (product R) at each point of time, indicating that product R does not inhibit phagocytosis of human monocytic cells. Other biological functions of the product R: Some of the other known biological functions of the product R have been described in U.S. Patent Nos. 5,807,840, 5,807,839 and 5,902,786; in U.S. Patent Applications Serial No. 08 / 838,077, 08 / 838,069, 08 / 835,793, 08 / 735,794, 08 / 833,950, 08 / 837,992, 08 / 837,988, 08 / 838,070, 08 / 834,190, 08 / 835,791, 08 / 838,134, 08 / 839,651, 0 / 835,796, 08 / 964,250, 08 / 964,427, 08 / 923,516, 08 / 923,343, 08 / 922,888. 09 / 189,172, 09 / 007,565, 09 / 316,624, 09 / 316,374, 09 / 257,739, and in the publication of Hirschman and co-authors, J. Investig. Med., 1996; 44: 347-351. These patents, patent applications and publications are incorporated herein by reference to them, in their entirety.

CONCLUSIONS It has thus been determined that the composition of the product R, prepared according to the methods described herein, comprises nucleotides and peptides having molecular weights not exceeding 14 KDa, primarily not exceeding 8 KDa. The peptide components of the product R are unequally distributed and are typically located in the two silver-stainable main bands, which have molecular weights of 4.3 KDa, 5.2 KDa and a band less than 7.6 KDa. The UV absorption spectrum of the product R typically shows a maximum absorption at 260 nm and a transit at 235 nm, and the characteristic ratios of the absorbance at 260 nm with respect to the absorbance at 280 nm is 1.998, and at 260 nm with respect to 230 nm is 1,359. The HPLC profile of the product R comprises fractions of A, B, C, D, E, F, G, H, I, J, K, L and M, as shown in Figure 2. The filtration profile in BioGel P-2 gel, of the product R, comprises the fractions I, II, IIa, IIb, III, Illa, IV and IVa which are shown in Figure 3.

COMPARISON BETWEEN THE CONVENTIONAL RETICULOSE® COMPOSITION AND THE PRODUCT R The composition of the product R prepared according to the teachings of the present invention is compared with the conventional composition of RETICULOSE®, with respect to its molecular weights (MW) and ultraviolet (UV) absorbances (A) at wavelengths of 230 nm, 260 nm and 280 nm, as shown in Table IV. Although it has been reported that components having molecular weights below 15 KDa of RETICULOSE® inhibit phagocytosis, the present application demonstrates that product R does not inhibit phagocytosis.

TABLE IV MW UV l / PH * »60 1280. 26030. Product R < 14 KDa 1,998 1,359 NO RETICULOSE® 1-25 KDa 2,839 1,198 Yes "Inhibition of phagocytosis by molecules having a molecular weight below 15 KDa.

Thus, the product R differs substantially from RETICULOSE® in its composition and in its biological functions. Table V is a comparison between the relative amounts of the starting materials used for the preparations of the present therapeutic composition, the product R, and the conventional composition RETICULOSE®.

TABLE V Starting materials for the initial reaction, in 10 liters.- RETICULOSE® Product R.

Casein 250 grams 140 grams Resin peptone 150 grams 68.4 grams Serum albumin 15 grams 13 grams RNA 80 grams 88 grams NaOH 75 grams 66 grams About 221 g of proteins are used in the initial reaction, for the preparation of the product R, while about 415 g is used for the preparation of RETICULOSE®. Therefore, the initial protein concentration for the RETICULOSE® preparation is twice that of the preparation of the product R. The following example serves only as an illustration of the process for preparing the product R, and should not be considered as a limitation to the present invention.

EXAMPLE About 35.0 g of casein, about 17.1 g of beef peptone, about 22.0 g of nucleic acid (yeast RNA), about 3.25 g of bovine serum albumin, in about 2.5 liters of water for injection are suspended. USP, at approximately 3 to 7 ° C, in a suitable container, and shake gently until it has been properly moistened. all the ingredients. Carefully add, while stirring, about 16.5 g of sodium hydroxide (quality of ACS reagent), and continue stirring until the sodium hydroxide is completely dissolved. It is autoclaved at approximately 4.08 kg of pressure and 93 ° to 110 ° C for a period of time, until the RNA is completely digested, for example, approximately 4 hours. At the end of the period, the treatment is stopped in an autoclave and the reaction flask and its contents are allowed to cool slowly, to room temperature. Then it cools for at least six hours at around 3-8 ° C. The resulting solution is filtered through 2 micron and 0.45 micron filters, using inert gas, such as nitrogen or argon, at low pressure (6.89 to 41.36 kPa). Similarly, the solution is filtered again through 0.2 micron filters for pyrogen retention. The resulting filtrate is sampled and analyzed for total nitrogen. A calculation is then made to determine the amount of cold water for injection to be added to the filtrate to produce a diluted filtrate with a nitrogen content of between about 165 and 210 mg / 100 mL; The final volume is approximately 5 liters. The pH is then adjusted with either concentrated HCl (reagent quality, ACS), or with normal 1.0 NaOH, approximately at the 7.3-7.6 scale. The diluted solution is then filtered through 0.2 miter filters with inert gas at low pressure. Filled with 2 ml glass ampoules and sealed while they are under gas atmosphere inert. The ampules are collected and treated in an autoclave for final sterilization at 115 ° C and pressure of 6.35-7.26 kg, for around 30 minutes. After the sterilization cycle, the ampoules are cooled and washed with the product R. All the quantities are subject to a variation of plus or minus 15 percent for the pH, the volume and the analytical adjustments. Thus, even though the fundamental novel aspects of the invention have been shown and described and indicated, as they are applied to a preferred embodiment thereof, it will be understood that those who are experts in the matter may make various omissions and substitutions and changes in the form and in the details of the devices illustrated, as well as in their operation, without departing from the spirit of the invention. For example, it is expressly intended that all combinations of those elements and / or method steps that perform substantially the same function, in substantially the same manner, to obtain the same results, are within the scope of the invention. Moreover, it must be recognized that the structures and / or elements and / or method steps shown and / or described in relation to any described form or modality of the invention, may be incorporated in any other form or manner described, exhibited. or suggested, as a general matter of design selection. Therefore, it is the intention that the only limitation is indicated by the scope of the claims that follow.

Claims (9)

1. - A peptide nucleic acid composition that absorbs light at wavelengths of 230 nm, 260 nm and 280 nm, so as to result in an absorption ratio at 260 nm / 280 nm of about 1,998, and a ratio of absorption at 260 nm / 230 nm of around 1359; comprising nucleotide molecules resulting from a plant RNA and peptides resulting from a mixture of casein, beef peptone and bovine serum albumin; said molecules having molecular weights distributed unevenly.

2. The composition according to claim 1, further characterized in that the nucleotides are mononucleotides. 3.- The composition in accordance with the claim 1, further characterized in that the molecules have molecular weights distributed non-uniformly, on a scale of zero to substantially no more than 14 KDa. 4. The composition according to claim 1, further characterized in that the molecules have molecular weights distributed in a non-uniform manner, on a scale from zero to substantially no more than 8 KDa. 5.- The composition in accordance with the claim 1, characterized in that the molecules have substantial concentrations at molecular weights of substantially 5.2 KDa and 4.3 KDa. 6. A method for preparing a peptide nucleic acid composition that absorbs light at wavelengths of 230 nm, 260 nm and 280 nm, so as to result in an absorption ratio of 260 nm / 280 nm around of 1998 and an absorption ratio of 260 nm / 230 nm of about 1,359; the composition containing nucleotide and peptide molecules having molecular weights unevenly distributed; characterized in said method because it comprises the steps of: a) forming a mixture that includes a combination of proteins consisting of casein, beef peptone and bovine serum albumin; a plant RNA and a base in water, where the ratio of the protein to water combination is on a scale of about 1.5 / 100 to about 2.5 / 100, by weight; b) processing the mixture at an elevated temperature and at elevated pressure, so as to form a solution and insoluble elements; c) eliminate the insoluble elements; d) dilute the solution in water; and e) after performing steps b, c and d, adjust the pH of the solution to a physiologically acceptable pH. 7. The method according to claim 6, further characterized in that the ratio of the protein to water combination is about 2.2 / 100 by weight. 8. The method according to claim 6, further characterized in that the nucleotides are mononucleotides. 9. The method according to claim 6, further characterized in that the molecules have non-uniform molecular weights, on a scale from zero to substantially no more than 14 KDa. The method according to claim 6, further characterized in that the molecules have non-uniformly distributing molecular weights, on a scale from zero to substantially no more than 8 KDa. 1. The method according to claim 6, further characterized in that the molecules have substantial concentrations at molecular weights of substantially 5.2 KDa and 4.3 KDa.